ABCA1 Gene Regulation: The ATP binding cassette transporter A1 (ABCA1) protein mediates efflux of cholesterol and phospholipids to poorly lipidated HDL from macrophages and other cells. By using site-directed mutagenesis to generate plasmids containing mutations in the E-box, we showed that mutation or deletion of the E-box motif caused a 3-fold increase in promoter transcriptional activity in RAW and 293 cells under basal conditions. This effect was further amplified by stimulation with 9-cis retinoic acid and oxysterols. Gel shift and DnaseI footprint analysis showed competition for DNA binding by 27bp fragments containing the wild type but not the mutant E-box and protection of the E-box from DnaseI digestion, indicative of a protein binding to this region. Preincubation of nuclear extracts with antibodies established that the E-box binding proteins, USF1 and USF2 as well as fos-related antigen 2 (Fra2) bind to the E-box in the human ABCA1 promoter. Co-transfection of USF1 and USF2 enhanced but Fra2 repressed ABCA1 promoter activity. Thus, a complex consisting of USF1, USF2 and Fra2 bind to the E-box motif 147 bp upstream of the transcriptional start site and facilitate repression of the human ABCA1 promoter. These combined studies identify a new mechanism mediating transcriptional repression of the human ABCA1 promoter.